cDNA Libraries and RNA Editing

(i) When constructing cDNA libraries it is very important to copy the whole of an mRNA into cDNA. One way to try and ensure that the 5' end of a mRNA is represented in a cDNA copy is to use "cap-trapper" technology. Explain in your own words and with a simple diagram how this works (do not include other steps in cDNA library construction- just what is relevant to explain "cap-trapper").

(ii) There is a type of RNA editing where a specific C residue in a mRNA is deaminated to produce a U residue. Only very few such examples are known, but perhaps there are more. Using mice as an example, how might you find other possible examples (that have not yet been discovered, or at least published) AND what would satisfy you as proof of RNA editing (where a C residue in a transcript was converted to a U residue)?

(iii) "Next-generation" or high-throughput sequencing (HTS) techniques currently allow many templates to be read in parallel but the amount of sequence derived from any one template is relatively short compared to dideoxy sequencing ("454" sequencing can produce up to about 500nt but other methods produce no more than 50nt [& compensate by producing millions of parallel reads]). These new sequence technologies might be used for sequencing the genomes of organisms for the first time or for looking at sequence variation among individuals where a prototype genomic sequence has already been established.
What do you think is the biggest difficulty in using an HTS technology with very short reads for these purposes, explaining as precisely as you can by including thoughts about (a) the type of organism and
(b) which type of application presents the biggest challenge?

(iv) HTS (above) can be applied to RNAs (called "RNASeq") by first ligating RNA to oligos and then converting RNAs to cDNA. In fact, because many HTS methods produce only short reads it is standard practice to begin by fragmenting RNAs (as for DNA in shotgun sequencing) so that a long mRNA can generate several fragments and a large number of starting points for sequence determination. The sequences of the original RNA molecules are then reconstructed just as contigs are constructed in shotgun sequencing. Remember that the RNAs are not purified individually- they are purified as a complex mixture from whole cell or tissue extracts. A recent article suggests that there are on average about seven alternative splicing events per multi-exon gene in humans.

Do you think RNASeq will contribute significantly to understanding the full variety of splicing events in human mRNAs AND what limitation does it have (just explain the single most important limitation that you can see)?

(v) If you (assuming you have the general resources of companies that can make various types of microarrays) want to make a microarray for measuring the patterns of gene expression in different cell types of an organism
(a) what do you need to know?
(b) do you need to have any specific cloned DNAs?

(vi) What factors limit the sensitivity of the kind of microarray study mentioned above? Make your answer a self-contained, short discussion of detection sensitivity that would be instructive for others to read.